Project Summary This proposal represents the continuation of our long-running program to understand the biology of polyamine metabolism in the trypanosomatid parasites and to exploit the pathway for drug discovery. Trypanosomatids are the causative agents of human African trypanosomiasis (HAT), Leishmaniasis and Chagas disease, all of which are listed by the WHO as neglected tropical diseases (NTDs). Collectively 18-20 million people are infected with one of these parasites, yet drug therapies remain inadequate for all three diseases. Polyamines are small organic polycations that are synthesized from L-ornithine and S-adenosylmethionine. In eukaryotes, the polyamine spermidine is absolutely essential for growth due to its role as a substrate for the hypusine modification of the translation factor eIF5A. Eflornithine, which inhibits the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), is a frontline therapy for the treatment of HAT. Interest in understanding the biology and regulatory mechanisms of this pathway led to the finding that, while the trypanosomatids share in common the essential role for hypusination, they have evolved both unique polyamine-containing metabolites and their own regulatory strategies in comparison to other eukaryotic cells. This includes our discovery that two of the biosynthetic enzymes S-adenosylmethionine decarboxylase (AdoMetDC) and deoxyhypusine synthase (DHS) each require oligomerization with inactive pseudoenzmes for activity. Despite significant understanding of the metabolism and biological roles of polyamines in trypanosomatids, a number of key questions remain unexplored and are the subject of this proposal. In Aim 1, we plan studies to build on our observation that the AdoMetDC pseudoenzyme (termed prozyme) is translationally regulated in response to perturbations that reduce levels of the product decarboxylated AdoMet, suggesting dcAdoMet functions as a novel metabolic signal for regulation of the polyamine pathway. In Aim 2, we will focus on unexplored metabolic pathways and functional roles of the upstream polyamine metabolites L-Orn and Put. Our questions are: does Put play additional roles beyond its requirement as a precursor for Spd synthesis; and can T. brucei synthesize L-Orn using an uncharacterized amidinotransferase. In Aim 3, we will study the function and composition of enzymes (DHS and deoxyhypusine hydroxylase (DOHH)) required to modify eIF5A with the hypusine cofactor. Our approaches will be a combination of genetic strategies and pharmacologic tools to perturb cell metabolism, state of the art metabolomics, genomic strategies to identify candidate genes, and biochemistry (protein expression, enzyme assay and crystallography). While our work will focus on T. brucei, the unusual metabolic features of this pathway have been found in all three pathogenic trypanosomatids, so it is likely that the discoveries we make in T. brucei will translate to T. cruzi and Leishmania. Finally, while our past grant also focused primarily on biological discovery, the results from our work laid the foundation for us to spin off two separate drug discovery programs targeting the trypanosomatids.